Composite Nanoparticle Structures for Chemical and Biological Sensing

ABSTRACT

Described herein is a nanoparticle that enhances the interaction of the nanoparticle and/or a molecule/material deposited on the surface of the nanoparticle with light, comprising a pair of stacked metallic disks separated by a non-metallic spacer, wherein: (a) the dimensions of the disks and spacer are smaller than the wavelength of the light; and (b) the nanoparticle enhance the light interaction at least three times greater than that an individual metallic disk. Methods for making the nanoparticle and methods for using the nanoparticle in a variety of assays are also provided.

CROSS-REFERENCING

This application is a continuation-in-part of U.S. application Ser. No. 13/838,600, filed Mar. 15, 2013 (NSNR-003), which application claims the benefit of U.S. provisional application Ser. No. 61/622,226 filed on Apr. 10, 2012, and is a continuation-in-part of U.S. patent application Ser. No. 13/699,270, filed on Jun. 13, 2013, which application is a § 371 filing of US2011/037455, filed on May 20, 2011, and claims the benefit of U.S. provisional application Ser. No. 61/347,178, filed on May 21, 2010;

this application is also a continuation-in-part of U.S. application Ser. No. 13/699,270, filed Jun. 13, 2013 (NSNR-001), which application is a § 371 filing of international application serial no. US2011/037455, filed on May 20, 2011, which application claims the benefit of U.S. Provisional Patent Application Ser. No. 61/347,178 filed on May 21, 2010; and

this application is also claims the benefit of: provisional application Ser. No. 61/801,424, filed Mar. 15, 2013 (NSNR-004PRV), provisional application Ser. No. 61/801,096, filed Mar. 15, 2013 (NSNR-005PRV), provisional application Ser. No. 61/800,915, filed Mar. 15, 2013 (NSNR-006PRV), provisional application Ser. No. 61/793,092, filed Mar. 15, 2013 (NSNR-008PRV), provisional Application Ser. No. 61/801,933, filed Mar. 15, 2013 (NSNR-009PRV), provisional Application Ser. No. 61/794,317, filed Mar. 15, 2013 (NSNR-010PRV), provisional application Ser. No. 61/802,020, filed Mar. 15, 2013 (NSNR-011PRV) and provisional application Ser. No. 61/802,223, filed Mar. 15, 2013 (NSNR-012PRV), all of which applications are incorporated by reference herein for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. FA9550-08-1-0222 awarded by the Defense Advanced Research Project Agency (DARPA). The government has certain rights in the invention.

BACKGROUND

There is a great need to develop new nanoparticle structures, and new fabrication methods for applications in chemical and biological sensing. The subject nanoparticle structures can greatly enhance (e.g. amplify) optical signals (particularly luminescence (e.g. fluorescence) and Surface Enhanced Raman Scattering (SERS)) disposed on or near to the nanoparticle surface; improve the detection sensitivity of the chemical and/or biological properties of the molecules disposed on or near the nanoparticles, improve nanoparticle performance in penetrating biological cells or materials, and in reducing biological toxicities. The subject fabrication methods allow the fabrication of such new nanostructures which are otherwise either impossible or hard to fabricate.

Conventional NPs, limited by their fabrication method (chemical synthesis), have simple architecture (spheres, rods, and shells), simple and smooth surfaces, and simple compositions (either pure dielectric, pure metal, or dielectric enclosed completely by metal (or vice versa)). All of these brought severe drawback to in-vivo diagnosis. The four major drawbacks are: (a) lower plasmonic enhancement than Au clusters or other plasmonic structures made on substrate, (b) large particle size for in-vivo diagnosis wavelength (e.g. 300 nm for Au NPs), (c) bio-undegradeable (for metal particles or nanoshells), and (d) similar surface property in entire surface rather surface location selective. They also have large particle size variations (˜15%). These drawbacks lead (i) poor in-vivo performances of low brightness (low enhancement in fluorescence or SERS), poor in-vivo suitability (slow and low number NPs entering cells), poor biocompability/safety (invasive and particle accumulation), and poor selectivity

In bio-safe in-vivo plasmonic-based diagnosis and therapeutics, one most significant roadblock is how to satisfy simultaneously two completely conflicting requirements on nanoparticle size: large (over 50 nm) for better therapeutic and diagnostic efficacy (i.e. plasmonic effectiveness and decent blood retention time), and ultra-small (sub 10 nm) for lower toxicity (i.e. rapid clearance from cell/body and hence zero accumulation).

Another major roadblock is that conventional nanoparticle fabrication methods prohibit current plasmonic nanoparticles from having the complex structures needed for achieving an ultra-high plasmonic enhancement that is several orders of magnitude higher than the current ones.

For examples, to have decent plasmonic effects and decent blood retention time, conventional approaches use gold spheres of 300 nm diameter, nanoshells of 60 nm diameter, and nanorods of 10 diameter and 60 nm long. These sizes are much larger than that for quick clear-up in cells or bodies, which should be sub-10 nm. Furthermore, for ultra-high plasmonic enhancements, it requires complex particle structures, such as nanogaps and nano-sharp-edges, which are missing in the current NPs, making them several orders of magnitude less plasmonic effective than what we are able to achieve (see section III). Clearly, the current NPs cannot simultaneously satisfy the size requirements for plasmonic effectiveness and bio-safety, because these nanoparticles are not biodegradable and hence cannot change from large size for plasmonic effectiveness to small size needed for bio clear-out.

In summary, all previous approaches cannot solve the major roadblocks of efficacy-safety conflict caused by conflicting particle-size requirements nor low plasmonic effects caused by lacking complexity in nanostructure.

Therefore, to advance diagnosis (in vitro and in-vivo) and single biological cell analysis, we need both new nanoparticle platforms (different architectures and physical principles) and new fabrication methods, that radically differ from conventional approaches. This is subject of current invention.

SUMMARY

The following brief summary is not intended to include all features and aspects of the present invention, nor does it imply that the invention must include all features and aspects discussed in this summary.

The invention is related to nanoparticle structures, fabrication methods and applications in chemical and biological sensing. The nanoparticles in the invention has very different structures and materials from the conventional metallic nanoparticles, which allows the new nanoparticle have many unique properties desired in sensing and diagnostics, including much more effective in enhancing a sensing light signal than the conventional metallic nanoparticles while having a size much smaller. The small sizes are important to in vivo testing and bio-safety. The unique structures of the nanoparticles cannot be made by conventional synthesis method, but are fabricated by template deposition and exfoliations. The nanoparticles in the invention also can biodegradable. In particular, the nanoparticle structures can greatly enhance (e.g. amplify) light absorption, light scattering, and light radiation, optical signals (particularly luminescence (e.g. fluorescence) and Surface Enhanced Raman Scattering (SERS)) disposed on or near to the nanoparticle surface (the nanoparticle can be inside biological cell and/or human body); improve the detection sensitivity of the chemical and/or biological properties of the molecules disposed on or near the nanoparticles, improve nanoparticle performance in penetrating biological cells or materials, and in reducing biological toxicities. The subject fabrication methods allow the fabrication of such new nanostructures which are otherwise either impossible or hard to fabricate. The functionalized nanoparticles can be used as biological and chemical assay for detection of biological and chemical markers (also termed “analytes”), such as proteins, DNAs, RNAs, and other organic and inorganic molecules, in single cells, tissue, and in-vivo for human and animals.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way. Some of the drawings are not in scale.

FIG. 1 Schematic of Stacked-nanoparticles (S-NPs): (a) a pair of metal-disks separated by a single dielectric disk; (b) S-NP with a magnetic disk on top; (c) S-NP with five disks; (d) 4 S-NPs glued by another dielectric material into a single particle.

FIG. 2 Schematic of (a) DS-NPs: S-NP with metal nano-dots self-assembled at the side wall, and (b) ES-NP (enhanced S-NP) with two non-metallic disks 460, each of them covers the exterior surface of the metallic disks of a S-NP.

FIG. 3 Biodegradation of stacked-nanoparticles (S-NPs).

FIG. 4 Schematics of coating a molecular adhesion layer and then the capture agent.

FIG. 5 schematically illustrates an exemplary antibody detection assay.

FIG. 6 schematically illustrates an exemplary nucleic acid detection assay.

FIG. 7 schematically illustrates another embodiment nucleic acid detection assay.

FIG. 8 shows a flow chart of Nano-PrinTED (nanoprint by templated exfoliatable deposition) and Dip-print of biodegradable dielectrics. Nano-PrinTED comprises three key steps: (i) have a template with nanostructured protrusions or hollows (e.g. dense nanopillar array on 4″ wafer), (ii) deposit a release layer (optional) and then multiple-layer composite materials to form nanoparticles on the pedestals of the template's protrusions or inside the hollows, and (iii) exfoliate the nanoparticles from the template by transfer printing or lift off. Dip-print is for patterning biodegradable dielectrics, since such materials cannot be thermally evaporated. Dip-print first puts a thin layer of liquid polymer precursors with proper viscosity on a plate (termed “material transfer plate”); and then presses the template used in Nano-PrinTED against the material transfer plate, picking up the polymer precursors only at the top of the pillars of the template. Afterwards the polymer precursors will be cured to form a solid polymer. The dip-printed polymers will be used for the dielectrics between the stacked layers and for the dielectrics that glue different columns into a single particle (Note different viscosity liquid will be used depending upon the gap size between the columns).

FIG. 9 Nano-PrinTED (nanoprint by templated exfoliateable deposition)—a new nanoparticle fabrication technology. Schematics of (a) Pillar template fabricated by lithography (e.g. NIL); (b) multiple deposition and self-assembly to form D2-particles; (c) transfer-print S-NPs to another substrate; and put in solution.

FIG. 10 Nano-PrinTED (nanoprint by templated exfoliateable deposition)—method 2. Schematics of (a) hole template (polymer) fabricated by lithography (e.g. NIL); (b) multiple deposition and self-assembly to form D2-particles inside the holes; (c) lift off the polymer (including the top stacked plane) around S-NPs; and put in solution.

FIG. 11. Dip-print of biodegradable dielectrics. Schematics of (a) pillar template fabricated by lithography (e.g. NIL); (b) deposition thin metal disks on top of the pillars; (c) Puts a thin layer of liquid polymer precursors with proper viscosity on a plate (termed “material transfer plate”); and then presses the template used in Nano-PrinTED against the material transfer plate, (d) picking up the polymer precursors only at the top of the pillars of the template. (e) add another deposition step to form top metal disk if needed and (f) put S-NPs into solutions.

FIG. 12. Dip-print of biodegradable dielectrics that “glue” columns. The same as dip printing the dielectric spacer, except that the viscosity of polymer precursors may be changed to make the polymer precursor fill the gaps between the columns. (a) 4 close pillar template with 4 S-NPs on top. (b) pick up the polymer precursors at the top of the S-NPs on the template, fill the gaps in between and glue the 4 S-NPs. (c) put into solutions.

FIG. 13 Scanning electron microscopy (SEMs) of (a) double-metal-disk and single dielectric (D-particle); (b) triple-metal (or magnetic) dielectric-nanoparticle (TS-NP); (c) D-particle after the “self-perfection by liquefaction” (SPEL) to change the shape of 2 metal disks; (d) D-particles array on the substrate after the template lift-off.

FIG. 14. Scanning electron microscopy (SEMs) of (a) D-particles array on the substrate after the transfer printing. (b) D-particles exfoliated into solution.

FIG. 15 Nano-PrinTED (nanoprint by templated exfoliateable deposition)—a new nanoparticle fabrication technology. Top row: Schematic. And bottom row: scanning electron microscope (SEM) of experimental results. (a) Pillar template fabricated by lithography (e.g. NIL); (b) Multiple deposition and self-assembly to form D2-particles; (c) transfer-print DPs to another substrate; (d) put in solution. (e-h), SEM images.

FIG. 16 Nano-PrinTED and Dip-print have far better precision in controlling the NP structure dimensions (including the size and shape of each individual components, their spacing, and final particle). (a) SEM picture of D2-P before release and (b) Measured size distribution. Measured size variation of D2-particle fabricated by Nano-PrinTED (<5%) is 3 fold less than AuNP manufactured by chemical synthesis (>15%).

FIG. 17 Measurements of extinction spectrum of D2-particles with SiO2 layer thickness from 5 nm to 30 nm and constant Au layer thicknesses of 20 nm. Plasmonic resonant peak wavelengths redshift with increasing SiO2 layer thickness.

FIG. 18 Simulation of the size of nanoparticles with different architectures required for the same resonant wavelength at 800 nm. It clearly shows that S-NP has much smaller particle sized than conventional metallic sphere and disks for a given resonant wavelength.

FIG. 19 (a) Measured Surface Enhanced Raman Spectroscopy (SERS) signal of BPE, and (b) fluorescence signal of IR-800 dye with single D2-particle and gold nanoparticle. A single D2-particle has a SERS/Fluorescence enhancement over 100/30 fold higher than a single gold nanoparticle of similar diameter.

Corresponding reference numerals indicate corresponding parts throughout the several figures of the drawings. It is to be understood that the drawings are for illustrating the concepts set forth in the present disclosure and are not to scale.

Before any embodiments of the invention are explained in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings.

Definitions

Before describing exemplary embodiments in greater detail, the following definitions are set forth to illustrate and define the meaning and scope of the terms used in the description.

The term “molecular adhesion layer” refers to a layer or multilayer of molecules of defined thickness that comprises an inner surface that is attached to the S-NP and an outer (exterior) surface can be bound to capture agents.

The term “capture agent-reactive group” refers to a moiety of chemical function in a molecule that is reactive with capture agents, i.e., can react with a moiety (e.g., a hydroxyl, sulfhydryl, carboxy or amine group) in a capture agent to produce a stable strong, e.g., covalent bond.

The term “capture agent” as used herein refers to an agent that binds to a target analyte through an interaction that is sufficient to permit the agent to bind and concentrate the target molecule from a heterogeneous mixture of different molecules. The binding interaction is typically mediated by an affinity region of the capture agent. Typical capture agents include any moiety that can specifically bind to a target analyte. Certain capture agents specifically bind a target molecule with a dissociation constant (K_(D)) of less than about 10⁻⁶ M (e.g., less than about 10⁻⁷M, less than about 10⁻⁸M, less than about 10⁻⁹M, less than about 10⁻¹⁰ M, less than about 10⁻¹¹ M, less than about 10⁻¹² M, to as low as 10⁻¹⁶ M) without significantly binding to other molecules. Exemplary capture agents include proteins (e.g., antibodies), and nucleic acids (e.g., oligonucleotides, DNA, RNA including aptamers).

The term “nanosensor” refers to a nanoparticle that is functionalize with a capture agent.

The terms “specific binding” and “selective binding” refer to the ability of a capture agent to preferentially bind to a particular target molecule that is present in a heterogeneous mixture of different target molecule. A specific or selective binding interaction will discriminate between desirable (e.g., active) and undesirable (e.g., inactive) target molecules in a sample, typically more than about 10 to 100-fold or more (e.g., more than about 1000- or 10,000-fold).

The term “protein” refers to a polymeric form of amino acids of any length, i.e. greater than 2 amino acids, greater than about 5 amino acids, greater than about 10 amino acids, greater than about 20 amino acids, greater than about 50 amino acids, greater than about 100 amino acids, greater than about 200 amino acids, greater than about 500 amino acids, greater than about 1000 amino acids, greater than about 2000 amino acids, usually not greater than about 10,000 amino acids, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; fusion proteins with detectable fusion partners, e.g., fusion proteins including as a fusion partner a fluorescent protein, β-galactosidase, luciferase, etc.; and the like. Also included by these terms are polypeptides that are post-translationally modified in a cell, e.g., glycosylated, cleaved, secreted, prenylated, carboxylated, phosphorylated, etc., and polypeptides with secondary or tertiary structure, and polypeptides that are strongly bound, e.g., covalently or non-covalently, to other moieties, e.g., other polypeptides, atoms, cofactors, etc.

The term “antibody” is intended to refer to an immunoglobulin or any fragment thereof, including single chain antibodies that are capable of antigen binding and phage display antibodies).

The term “nucleic acid” and “polynucleotide” are used interchangeably herein to describe a polymer of any length composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides, or compounds produced synthetically (e.g., PNA as described in U.S. Pat. No. 5,948,902 and the references cited therein) which can hybridize with naturally occurring nucleic acids in a sequence specific manner analogous to that of two naturally occurring nucleic acids, e.g., can participate in Watson-Crick base pairing interactions.

The term “complementary” as used herein refers to a nucleotide sequence that base-pairs by hydrogen bonds to a target nucleic acid of interest. In the canonical Watson-Crick base pairing, adenine (A) forms a base pair with thymine (T), as does guanine (G) with cytosine (C) in DNA. In RNA, thymine is replaced by uracil (U). As such, A is complementary to T and G is complementary to C. Typically, “complementary” refers to a nucleotide sequence that is fully complementary to a target of interest such that every nucleotide in the sequence is complementary to every nucleotide in the target nucleic acid in the corresponding positions. When a nucleotide sequence is not fully complementary (100% complementary) to a non-target sequence but still may base pair to the non-target sequence due to complementarity of certain stretches of nucleotide sequence to the non-target sequence, percent complementarily may be calculated to assess the possibility of a non-specific (off-target) binding. In general, a complementary of 50% or less does not lead to non-specific binding. In addition, a complementary of 70% or less may not lead to non-specific binding under stringent hybridization conditions.

The terms “ribonucleic acid” and “RNA” as used herein mean a polymer composed of ribonucleotides.

The terms “deoxyribonucleic acid” and “DNA” as used herein mean a polymer composed of deoxyribonucleotides.

The term “oligonucleotide” as used herein denotes single stranded nucleotide multimers of from about 10 to 200 nucleotides and up to 300 nucleotides in length, or longer, e.g., up to 500 nt in length or longer. Oligonucleotides may be synthetic and, in certain embodiments, are less than 300 nucleotides in length.

The term “attaching” as used herein refers to the strong, e.g, covalent or non-covalent, bond joining of one molecule to another.

The term “surface attached” as used herein refers to a molecule that is strongly attached to a surface.

The term “sample” as used herein relates to a material or mixture of materials containing one or more analytes of interest. In particular embodiments, the sample may be obtained from a biological sample such as cells, tissues, bodily fluids, and stool. Bodily fluids of interest include but are not limited to, amniotic fluid, aqueous humour, vitreous humour, blood (e.g., whole blood, fractionated blood, plasma, serum, etc.), breast milk, cerebrospinal fluid (CSF), cerumen (earwax), chyle, chime, endolymph, perilymph, feces, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, sweat, synovial fluid, tears, vomit, urine and exhaled condensate. In particular embodiments, a sample may be obtained from a subject, e.g., a human, and it may be processed prior to use in the subject assay. For example, prior to analysis, the protein/nucleic acid may be extracted from a tissue sample prior to use, methods for which are known. In particular embodiments, the sample may be a clinical sample, e.g., a sample collected from a patient.

The term “analyte” refers to a molecule (e.g., a protein, nucleic acid, or other molecule) that can be bound by a capture agent and detected.

The term “assaying” refers to testing a sample to detect the presence and/or abundance of an analyte.

As used herein, the terms “determining,” “measuring,” and “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations.

As used herein, the term “light-emitting label” refers to a label that can emit light when under an external excitation. This can be luminescence. Fluorescent labels (which include dye molecules or quantum dots), and luminescent labels (e.g., electro- or chemi-luminescent labels) are types of light-emitting label. The external excitation is light (photons) for fluorescence, electrical current for electroluminescence and chemical reaction for chemi-luminscence. An external excitation can be a combination of the above.

The phrase “labeled analyte” refers to an analyte that is detectably labeled with a light emitting label such that the analyte can be detected by assessing the presence of the label. A labeled analyte may be labeled directly (i.e., the analyte itself may be directly conjugated to a label, e.g., via a strong bond, e.g., a covalent or non-covalent bond), or a labeled analyte may be labeled indirectly (i.e., the analyte is bound by a secondary capture agent that is directly labeled).

The term “hybridization” refers to the specific binding of a nucleic acid to a complementary nucleic acid via Watson-Crick base pairing. Accordingly, the term “in situ hybridization” refers to specific binding of a nucleic acid to a metaphase or interphase chromosome.

The terms “hybridizing” and “binding”, with respect to nucleic acids, are used interchangeably.

The term “capture agent/analyte complex” is a complex that results from the specific binding of a capture agent with an analyte. A capture agent and an analyte for the capture agent will usually specifically bind to each other under “specific binding conditions” or “conditions suitable for specific binding”, where such conditions are those conditions (in terms of salt concentration, pH, detergent, protein concentration, temperature, etc.) which allow for binding to occur between capture agents and analytes to bind in solution. Such conditions, particularly with respect to antibodies and their antigens and nucleic acid hybridization are well known in the art (see, e.g., Harlow and Lane (Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989) and Ausubel, et al, Short Protocols in Molecular Biology, 5th ed., Wiley & Sons, 2002).

The term “specific binding conditions” as used herein refers to conditions that produce nucleic acid duplexes or protein/protein (e.g., antibody/antigen) complexes that contain pairs of molecules that specifically bind to one another, while, at the same time, disfavor to the formation of complexes between molecules that do not specifically bind to one another. Specific binding conditions are the summation or combination (totality) of both hybridization and wash conditions, and may include a wash and blocking steps, if necessary.

For nucleic acid hybridization, specific binding conditions can be achieved by incubation at 42° C. in a solution: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 65° C.

For binding of an antibody to an antigen, specific binding conditions can be achieved by blocking a substrate containing antibodies in blocking solution (e.g., PBS with 3% BSA or non-fat milk), followed by incubation with a sample containing analytes in diluted blocking buffer. After this incubation, the substrate is washed in washing solution (e.g. PBS+TWEEN 20) and incubated with a secondary capture antibody (detection antibody, which recognizes a second site in the antigen). The secondary capture antibody may conjugated with an optical detectable label, e.g., a fluorophore such as IRDye800CW, Alexa 790, Dylight 800. After another wash, the presence of the bound secondary capture antibody may be detected. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise.

The term “a secondary capture agent” which can also be referred to as a “detection agent” refers a group of biomolecules or chemical compounds that have highly specific affinity to the antigen. The secondary capture agent can be strongly linked to an optical detectable label, e.g., enzyme, fluorescence label, or can itself be detected by another detection agent that is linked to an optical detectable label through bioconjugatio (Hermanson, “Bioconjugate Techniques” Academic Press, 2nd Ed., 2008).

The term “biotin moiety” refers to an affinity agent that includes biotin or a biotin analogue such as desthiobiotin, oxybiotin, 2′-iminobiotin, diaminobiotin, biotin sulfoxide, biocytin, etc. Biotin moieties bind to streptavidin with an affinity of at least 10-8M. A biotin affinity agent may also include a linker, e.g., -LC-biotin, -LC-LC-Biotin, -SLC-Biotin or -PEGn-Biotin where n is 3-12.

The term “streptavidin” refers to both streptavidin and avidin, as well as any variants thereof that bind to biotin with high affinity.

The term “marker” refers to an analyte whose presence or abundance in a biological sample is correlated with a disease or condition.

The term “bond” includes covalent and non-covalent bonds, including hydrogen bonds, ionic bonds and bonds produced by van der Waal forces.

The term “amplify” refers to an increase in the magnitude of a signal, e.g., at least a 10-fold increase, at least a 100-fold increase at least a 1,000-fold increase, at least a 10,000-fold increase, or at least a 100,000-fold increase in a signal.

The term “local” refers to “at a location”,

Other specific binding conditions are known in the art and may also be employed herein.

It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise, e.g., when the word “single” is used. For example, reference to “an analyte” includes a single analyte and multiple analytes, reference to “a capture agent” includes a single capture agent and multiple capture agents, and reference to “a detection agent” includes a single detection agent and multiple detection agents.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

The following detailed description illustrates some embodiments of the invention by way of example and not by way of limitation.

The invention is related to nanoparticle structures, fabrication methods and applications in chemical and biological sensing. The nanoparticles in the invention has very different structures and materials from the conventional metallic nanoparticles, which allow the new nanoparticle having many unique properties desired in sensing and diagnostics, including much more effective in enhancing a sensing light signal than the conventional metallic nanoparticles while having a size much smaller. The small sizes are important to in vivo testing and bio-safety. The unique structures of the nanoparticles cannot be made by conventional synthesis method, but are fabricated by template deposition and exfoliations. The nanoparticles in the invention also can be biodegradable. In particular, the nanoparticle structures can greatly enhance (e.g. amplify) light absorption, light scattering, and light radiation, optical signals (particularly luminescence (e.g. fluorescence) and Surface Enhanced Raman Scattering (SERS)) disposed on or near to the nanoparticle surface (the nanoparticle can be inside biological cell and/or human body); improve the detection sensitivity of the chemical and/or biological properties of the molecules disposed on or near the nanoparticles (e.g. proteins, DNAs, RNAs, and other organic and inorganic molecules), improve nanoparticle performance in penetrating biological cells or materials, and in reducing biological toxicities. The subject fabrication methods allow the fabrication of such new nanostructures which are otherwise either impossible or hard to fabricate.

The invention covers four areas: (1) nanoparticle structures, (2) fabrication methods, (3) surface functioning, and (4) applications in chemical and biological sensing.

Certain physical principles and certain materials, dimensions gaps used in the invention are similar to “disk-coupled dots-on-pillar antenna array” (D2PA) which is on a solid support (as described in WO2012/024006 and WO2013154770 which are incorporated by reference).

Nanoparticle Structures and Materials Basic Structure

In one embodiment of the invention, as illustrated in FIG. 1, a nanoparticle 100, termed stacked-nanoparticle (S-NP), that enhances the interaction of the nanoparticle and/or a molecule/materials deposited on the surface of the nanoparticle with light, comprises at least a pair of stacked metallic disks 110 and 120, separated by a non-metallic spacer 130, wherein (a) the dimensions of the disks and spacer are smaller than the wavelength of the light; and (b) the nanoparticle enhances the light interaction at least three times greater than that of each said metallic disk. The metallic disks 110 and 120 can be made of the same or different materials and have the same or different thickness. Furthermore, a thin adhesion layer may be between two adjacent disks.

The non-metallic spacer 130 can be biodegradable, namely, it can be dissolved in a bio-environment. As illustrated in FIG. 3, when the spacer is degraded, a S-NP breaks into small pieces. Breaking up into small pieces has advantages in in vivo application; small pieces can come out biological cells and human body much faster than large pieces, hence avoiding accumulation. Since the space is not completely enclosed by the metallic disks (non-biodegradable), fluid can access the spacer from the side of the spacer. The biodegradation time can be controlled by the biodegradable material and their dimensions.

The light interaction include light absorption, light scattering, light radiation, Raman scattering, chromaticity, and luminescence that includes fluorescence, electroluminescence, chemiluminescence, and electrochemiluminescence.

A preferred wavelength range for the light that can be enhanced by the S-NP is about from 20 nm to 10 micrometer. Another preferred the wavelength range is from 300 nm to 4000 nm. For in vivo applications, a preferred wavelength range (window) for a good light penetration in biological tissue is about from 630 nm to 1316 nm.

The molecule/materials deposited on the S-NP include the molecules/materials to be sensed, such as the analytes and/or their labels including proteins, DNAs, RNAs, and other organic and inorganic molecules in single cells, tissue, and in-vivo for human and animals.

The “metallic” in this invention means that for a given light wavelength the electrons in the materials can generate plasmons. For example, the gold has a plasmon wavelength about 560 nm; for the wavelength longer than 560 nm, the gold behavior like “metallic”, for the wavelength significantly shorter than 560 nm, the gold behavior like a non-metallic to the light.

The sensing property includes the sensing signal intensity, sensing signal spectrum, limit of detection, detection dynamic range, and signal variation reduction (smaller error bar) of the sensing. The sensing includes the detection of the existence, quantification of the concentration, and determination of the states of the targeted analyte. The analyte includes proteins, DNAs, RNAs, and other organic and inorganic molecules. The invention can be used in the sensing in vitro, or in vivo.

The key reason for S-NP superior to the conventional NPs in enhancement with smaller size is due to different physics. Conventional NPs with metal all around follows the parabolic function or the dispersion relation

${{\frac{k_{x}^{2}}{ɛ_{1}} + \frac{k_{y}^{2}}{ɛ_{2}}} = \frac{\omega^{2}}{c^{2}}},$

where ε₁ and ε₂ are dielectric constants (permittivities) in two orthogonal directions and for conventional NPs, they both have the same sign. Hence, their isofrequency curve is elliptical, which leads to bounded wavevectors, k, and relatively long wavelength. But for S-NP with bipolar-permittivity (let ε₁=ε_(p) and ε₂=−ε_(v), having opposite sign, while ε_(p) and ε_(v) are positive values), the equation becomes

${{\frac{k_{v}^{2}}{ɛ_{p}} - \frac{k_{p}^{2}}{ɛ_{v}}} = \frac{\omega^{2}}{c^{2}}},$

a hyperbolic curve, which means that wavevector, k, in both directions is unbounded, and can be very large. This allows a very short wavelength inside the particle (outside the particle the wavelength is still 800 nm), and consequently a very small particle size for a large optical signal. Furthermore, a pair of disks forms a resonant cavity for the light.

As shown in FIG. 4, when the surface of the S-NP 100 is functionalized, it becomes a nanosensor 200 for sensing an analyte in biological and chemical detection. The surface functionalization can be many ways as discussed later, including attaching the capture agents that selectively bond to a targeted analyte. The analytes include proteins, peptides, DNA, RNA, nucleic acid, small molecules, cells, nanoparticles with different shapes. The targeted analyte can be either in a solution or in air or gas phase. The sensing includes light absorption, light scattering, light radiation, Raman scattering, chromaticity, luminescence that includes fluorescence, electroluminescence, chemiluminescence, and electrochemiluminescence. The sensing property includes the sensing signal intensity, sensing signal spectrum, limit of detection, detection dynamic range, and signal variation reduction (smaller error bar) of the sensing. The sensing includes the detection of the existence, quantification of the concentration, and determination of the states of the targeted analyte. The invention can be used in the sensing in vitro, or in vivo.

NP Structure Variations and Improvements

Dots-On-Sidewall S-NP (DS-SP).

One embodiment of S-NP (FIG. 1b ), termed “dots-on-sidewall S-NP” (DS-SP) 400 comprises metallic nanodots 440 on the sidewall of the spacer and/or the metallic disks, which can have a higher light signal enhancement than the S-NP without them. For 800 nm wavelength, the diameter of the nanodots is about 3 to 15 nm.

Magnetic/Magnetizable S-NP (MS-NS).

S—NP can be made to be magnetic/magnetizable, so that they can be attracted to a magnet, and is termed MS-NP. One embodiment of MS-NP comprises a S-NP having at least one magnetic/magnetizamble disk 140 stacked on a normal S-NP, as shown in FIG. 1 b.

Enhanced S-NP (ES-NP).

The S-NP light signal enhancement can be further increased. For a S-NP, the light signal enhancement on its surface is not uniform: the regions with sharp (i.e. small curvature) edges of metallic materials and the small gaps between two metallic materials are the high enhancement regions, while the other regions are low enhancement regions (e.g. the flat exterior surface of the metallic disk). The high enhancement region means that a molecule or a material attached to that region will have its light signal amplified more than that of attaching to a low enhancement region. For example, a fluorophore (e.g. a fluorescence molecule) attached to the high enhancement region will have a higher fluorescence signal under a light excitation than that to attached a low enhancement region.

One embodiment of the invention is to selectively mask the low enhancement regions from a molecular binding and selectively attach the molecules that its light signal will be amplified (e.g. the capture agent that binds an analyte with a light label) to the high enhancement regions. One way to achieve this is to add two non-metallic disks as masking disks, one on each side of the S-NP, to mask the exterior surface of the metallic disks of a S-NP from the attachment of a molecule, as illustrated 150 and 160 in FIG. 1c , and 460 in FIG. 2. Such particle 400 is termed ES-NP (enhanced S-NP), which has five disks (2 metallic disks and 3 non-metallic disks). For example, a 3 nm thick of SiO2 disk can be used as the masking disks and the molecules are DSU, which only attach to the metal. Of course, one chose to mask one-side of disk or both size. The masking disks may use different thickness and different materials. For some applications, a part of disk edges are also masked. Some other details have be disclosed in PCT/US14/29979, filed on Mar. 15, 2014, and 61/801,424, filed on Mar. 15, 2013, which are incorporated by reference herein.

S-NP with More Stacked Metallic Disks.

In certain embodiments, S-NP has more than three disks; it can have 4, 5, 6, or more disks as much as required in sensing. The metallic disks also can be more a pair.

Bundled S-NPs (BS-NP) (S-NP with Multi-Columns).

One embodiment of S-NP is to pack several S-NPs together into one larger particle (a bundle) using a biodegradable dielectric material glue (FIG. 1.D). The reason is that such bundle allows the bundle having the same or similar light signal enhancement as a single S-NP with the same size, but after biodegradable, the bundle has much smaller pieces and hence easy to be cleared from biological cells and human body.

Disks Shape and Dimension

The lateral shapes of the disks of S-NP can be selected from the shape of round, square, rectangle, polygon, elliptical, elongated bar, polygon, other similar shapes or combinations thereof. In general, each disk can have different lateral shapes and dimensions from the others. In certain cases, as discussed later, one way to fabricate the S-NP is by using a template, deposition, exfoliation; such fabrication leads to similar lateral shape and dimensions for all disks in a given S-NP. But by using different templates each S-NP can have may different lateral shapes.

The shape of the top and bottom surface of the disks can be different, and can be flat, but also can be bulged, or a half-sphere. An example of the fabricated S-NP for 800 nm light wavelength are given in FIGS. 13, 14, and 15.

Dimensions for the metallic disks of S-NP and the spacer should be less than the wavelength of the light that the S-NP enhances. For a given wavelength, the light enhancement depends on the S-NP size and a resonant peak (vs. the size). The spacer between the pair of metallic disk has a thickness of 0.3 nm to 50 nm. This spacer's thickness has an important role in determine gap. In general smaller the gap is better, but a small gap also changes the resonant wavelength.

The disk diameters are often decided by balancing the light signal enhancement and the other requirements in in vivo application. For an example, to have an easy bio-cell and bio-material penetration and exit and bio-safety, the particle size should be as small as possible, but if the particle size is too small, it reduces the light enhancement factor. One embody of the invention is the S-NP that optimize both requirements.

As an example of DS-NP for 800 nm wavelength, the disks have a round shape of diameter from 30 nm to 100 nm, the top metallic disk thickness is from 5 nm to 30 nm, the spacer thickness is from 2 to 30 nm, and the bottom metallic disk thickness is from 5 nm to 30 nm, the self-assembled dots diameter is 5 nm to 15 nm, the magnetic disk thickness is from 5 to 30 nm, and the adhesion layers between the disks are titanium or Cr of a thickness from 0.5 nm-1 nm. Examples of the fabricated disks are shown in FIG. 13-16.

In a preferred DS-NP structure with light resonance absorption around 800 nm wavelength, the disks have a round shape of diameter of 70 nm, the top metallic (gold) disk thickness is 15 nm, the spacer (silicon dioxide) thickness is 20 nm, and the bottom metallic (gold) disk thickness is 15 nm, the self-assembled gold dots diameter is around 10 nm, and the adhesion layers between the disks are titanium of a thickness of 0.5 nm.

Metallic Materials

The materials used for the metal components (e.g. disk and dots) in S-NPs are chosen from materials that are metallic in the working photon wavelength. For examples, the metallic materials can be selected from gold, copper, silver, aluminum, their mixture, alloys, and multilayers in visible light ranges and longer wavelength, and certain metal oxides (as ITO, zinc oxide), for near or mid infrared wavelength or longer wavelength, or semiconductor (as silicon or gallium arsenide) for certain wavelength range. One can use a single material or a combination of them.

Materials for Non-Metallic Spacer.

The materials for the non-metallic spacer and non-metallic masking layers in S-NPs are chosen from dielectric materials and/or semiconductors. The material can be bio-degradable or non-bio-degradable. One important condition in selecting these materials is their effects on the light enhancement of S-NP. In many embodiments, such enhancement should be as high as allowed.

The dielectric materials can be inorganic or organic either in crystal, polycrystalline, amorphous, or hetero-mixture, and combination of one or more thereof depends on the applications. For examples, inorganic dielectric components can be selected from silicon dioxide, diamond, graphite, titanium dioxide, other certain metal oxides, and inorganic compounds in light wavelength range smaller than their energy bandgap. Organic dielectric components can be selected from polymers as biodegradable polymers (list before) for certain applications, other polymer as biopolymer (e.g. polynucleotides, cellulose), copolymer (e.g. styrene-isoprene-styrene), conductive polymer (e.g. poly(p-phenylene vinylene)), fluoropolymer, polyterpene, phenolic resin, polyanhydrides, polyester, polyolefin, rubber, superabsorbent polymer, vinyl polymer, etc.; or from small molecules, e.g. fullerene derivative, benzene derivatives, etc. The semiconductor materials can be inorganic or organic either in crystal, polycrystalline, amorphous, or hetero-mixture, and combination of one or more thereof depends on the applications. One can use a single material or a combination of them.

Biodegradable Materials

The biodegradable polymers for S-NPs are a specific type of polymers that is stable and durable enough for use in their intended applications and easily breaks down (to form gases, salts, or biomass) after its degradation.

These biodegradable polymers contains two major types: agro-polymers (derived from biomass, e.g. poly(saccharide)s as the starches in wood, cellulose, chitosan, proteins), and biopolyesters (derived from micro-organisms or synthetically made from either naturally or synthetic monomers, e.g. polyhydroxybutyrate and polylactic acids). Most of biodegradable polymers consist of ester, amide, or ether bonds. More examples of these biodegradable polymers contains: poly(esters) based on polylactide (PLA), polyglycolide (PGA), polycaprolactone (PCL), and their copolymers, poly(hydroxyalkanoate)s of the PHB-PHV class, other hydrolytically degradable polymers (as polyurethanes, poly(ester amide), poly(ortho esters), polyanhydrides, poly(anhydride-co-imide), pseudo poly(amino acide), poly(alkyl cyanoacrylates), etc.), other enzymatically degradable polymers (proteins (as collagen, elastion), poly(amino acids), polysaccharides, etc), and other natural polymers. One can use a single material or a combination of them.

Magnetic/Magnetizable Materials

The magnetic/magnetizable materials are those materials that will experience a magnetic force in a magnetic field. The can be selected from: ferromagnetic (e.g. iron, cobalt, nickel, some of the rare earths (gadolinium, dysprosium), etc.), ferrimagnetic (e.g. iron(II,III) oxide, yttrium iron garnet, cubic ferrites composed of iron oxides and other elements such as aluminum, cobalt, nickel, manganese and zinc, hexagonal ferrites, and pyrrhotite, etc.), superparamagnetic, and other suitable magnetic materials include oxides, e.g. ferrites, perovskites, chromites and magnetoplumbites, a rare earth/cobalt alloy or any other inorganic/organic compound with ferromagnetic/ferrimagnetic properties. One can use a single material or a combination of them.

Some key advantages are: the S-NPs have the right (large) particle size and complex shape for high-performance in-vivo plasmonic enhanced diagnostics and therapeutics, yet biodegradable, afterwards (with controlled timing), into sub-10-nm particles with a volume only 10% to 1% of original S-NP for quick cell/body clearance. Furthermore, S-NPs offer a plasmonic enhancement several orders of magnitudes higher than all current NPs.

To design different biocompatible and biodegradable S-particles that are not only effective in in-vivo therapeutic and diagnosis but also bio-safe, we need to control different S-NP's architectures, materials, dimensions and shapes.

1. Particle Size Requirements for Efficacy of Plasmonic-Based In-Vivo Therapeutic and Diagnosis.

Two factors that determine the sizes: (a) effectiveness of plasmonic effects and (b) the blood retention time—the time needed for sufficient circulation in blood to deliver proper dosage of nanoparticles to the specific sites.

To be plasmonic effective needs two things: first, the nanoparticle size has to be in resonance with in coming wavelength; and second, there should be small gaps and sharp edges. For the first plasmonic requirement at the in vivo penetration light wavelength of 800 nm (the entire window is NIR (670 to 890 nm)), for example, conventional approaches use gold spheres of 300 nm diameter, gold nanoshells of 60 nm diameter, and gold nanorods of 10 diameter and 60 nm long for decent plasmonic effects and decent blood retention time. The size smaller than those above will make these particles plasmonic extremely ineffective.

For the second plasmonic requirement, the conventional particles do not have the complex structures (nanogaps and nano-edges), and hence are much worse (orders of magnitude worse) than those that have such structures, which has been proofed by our experiment with S-particles.

To have sufficient blood retention time, the NP's size should be ˜50 to 200 nm range, and cannot be too small either. Too small particle size will make the particle cleared-out from cells and body quickly (see below), hence failing to deliver proper dosage, unless large nanoparticle dosage or repeated dosage of nanoparticles are intravenously administered, which will become unsafe and cause immunogenic response.

2. Particle Size Requirements for Bio-Safety.

Two nanoparticle sizes are very important in bio-safety in in-vivo diagnosis: (a) safe-particle-delivery size, which is the nanoparticle size that can be safely delivered to the specific sites without causing immunogenic response or any toxic effects, assuming low NP dose and no NP accumulation, and (b) particle clearance size, where NPs with such size can be easily cleared out from cells/body. As shown in FIG. 11, the particle safe-particle-delivery size has several bands, and the particle clearance size should be <10 nm (<6 nm even better).

3. Conflict in Particle Size Requirements.

Clearly, the size requirements are conflicting. For conventional plasmonic particles, which cannot change their size once are put in vivo, only way to solve this size requirement conflict is to compromise both the efficacy and the bio-safety to pick a particle size in the middle to balance plasmonic enhancement and the circulation within the body with the nanoparticles' ability to escape from the body.

The exact dimensions of these components depend on the light wavelength and materials. In some cases particular for the light wavelength of −800 nm and gold for the metallic materials and silicon dioxide as the dielectrics, the dimensions of the components can be in the range of 4 nm to 1500 nm and a thickness of the disks may be from 1 nm to 500 nm, depending on the exact wavelength of the light to be used in sensing. The gaps between components (e.g., the gaps between the metallic disks) may be in the range of 0.5 nm to 200 nm. For many applications, a small gap (in the range of 5 nm to 50 nm) may be used to enhance the optical signals.

This is one of the most challenging issues in plasmonic-based particle in vivo. Previous approaches have very limited success. For example, nanoparticle such as PEG-passivated gold nanoparticles, Nanoshells and gold nanorods. These nanoparticles employs surface coating or exotic shapes to achieve optimum optical resonance while retain a proper size (<100 nm) throughout the needed blood retention time. However, their ability of tuning the optical responses is still limited, thus the optical field enhancement is still weak. Moreover, these particles have a size much larger than the clearance size and hence will be accumulated in the body, becoming toxic.

S-NP Fabrication Methods Nano-PrinTED-1 (Protrusion Template).

As shown in FIGS. 8 and 9, one embodiment of the fabrication method, termed Nano-PrinTED using a protrusion template 1010, comprising three key steps: (i) have a template with nanostructured protrusions 1010 (e.g. dense nanopillar array on 4″ wafer (each pillar of the same or similar diameter selected from 5 nm to 100 nm), (ii) deposit a release layer (optional) and then deposit multiple-layers of materials 1020 needed to form nanoparticles on both the pedestals of the template's protrusions (each nanoparticle size is determined by the diameter of the template nanopillar) and inside the trenches, and (iii) exfoliate the nanoparticles 100 from the template.

Solvent Dissolve/Ultra-Sonic Exfoliation.

The template with S-NPs is put inside the container with particular solvent to dissolve the release layer under the S-NPs. The container can be put in an ultrasonicator to speed up the exfoliation process. S-NPs will be exfoliated in the solvent.

The exfoliation can be done in several ways: (1) Transfer printing exfoliation: The template with S-NPs is printed onto another substrate with a thin adhesion layer 1030 (e.g. certain polymer thin film). The larger adhesion force between the particles and the adhesion layer exfoliate all the S-NPs onto the new substrate. The S-NPs can be further released into solution by dissolving the adhesion layer on the new substrate similar to previous method. (2) Spin-on peel-off exfoliation: A kind of adhesion thin film (e.g. certain polymer) is spinned onto the template with S-NPs. After the curing of the film, the S-NPs are adhered in the adhesion layer, following a peel-off process to exfoliate all the S-NPs. The S-NPs can be further released into solution by dissolving the adhesion film as previous method. And (3) Wash exfoliation: The template with S-NPs are tilted above a beaker (or other container), and washed with certain solution (with spray gun). With the force of turbulent solution, the S-NPs will be exfoliated from the template and into the solution.

In certain embodiments, the same metal deposition for 1020 also form the nanodots on the disk sidewall to form S-NP, due to the fact that a thin metal on the sidewall self-assembled into nanodots, as shown from the experimental results in FIGS. 13, 14 and 15.

In the deposition (ii), the deposition uses a beamed materials (i.e. the material is deposited in one direction not in the other directions) and in the angle substantially normal to the template surface. Due the height of the pillars, the materials deposited in on the foot of the pillars are not connected to the materials deposited on the top of the pillar (i.e. pillars' pedestals), making the materials deposited on the pedestals forming S-NPs. The exfoliation free these S-NP from the template. The template can be used repeatedly until the trenches between the pillars are filled with material. When that happens, a cleaning step can be used to remove the deposited materials, and then the template can be reused again.

Nano-PrinTED-2 (Concave Template).

As shown in FIG. 10, one embodiment of the fabrication method, termed Nano-PrinTED using a concave template, comprising three key steps: (i) have a template with nanostructured wells 1100, (ii) deposit a release layer (optional) and then deposit multiple-layers of materials 1120 needed to form nanoparticles in the wells of the template's protrusions (each nanoparticle size is determined by the diameter of the well), and (iii) exfoliate the nanoparticles 100 from the template. Due the depth of the wells, the materials deposited on the bottom of the wells are not connected to the materials deposited on the top surface of the template, making the materials deposited inside well forming S-NPs.

All the templates can be in the form of a plate, a roller or roll or sheet, and can be in regard material or flexible. The template can be fabricated by nanoprint. The template materials can be any materials that mechanical strong enough for templating and chemical inner enough.

Methods of Deposition.

The methods to shadow deposit materials can be any method, as long as it is more or less directional, and can evaporate the intended materials. The deposition methods include evaporation, sputtering and chemical or molecular beams. The evaporation further includes the evaporation by chemical vapors, molecular beams, electron beam heating thermal heating, laser heating, and other heating methods. The sputtering includes the sputtering by ion, electron, plasmon, photon (i.e. laser), and other energetic particles.

Dip-Print.

The fabrication method of Dip-print is for patterning biodegradable dielectrics, since such materials cannot be thermally evaporated. The fabrication method of Dip-print, as shown in FIG. 12, first puts a thin layer of liquid polymer precursors with proper viscosity on a plate (termed “material transfer plate”); and then presses the template used in Nano-PrinTED against the material transfer plate, picking up the polymer precursors only at the top of the pillars of the template. Afterwards the polymer precursors will be cured to form a solid polymer. The dip-printed polymers will be used for the dielectrics between the stacked layers and for the dielectrics that glue different columns into a single particle (Note different viscosity liquid will be used depending upon the gap size between the columns).

The key advantages of this new fabrication technique are (a) form the complex structures that are needed for enhance plasmonic effects but cannot be formed in conventional fabrication methods, (b) have far better precision in controlling the NP structure dimensions (including the size and shape of each individual components, their spacing, and final particle), (c) a new way to solve the problem in patterning biodegradable materials, and (d) scalable to large volume and low-cost (e.g. As having demonstrated, the fabrication rate is 2×10̂11 particles per 4″ wafer per run, and it can be scaled up by over three orders of magnitude in throughput by roll-to-roll technology.) (ii) Fabrication: to advance new nanoparticle fabrication methods, nano-PrinTED and Dip-print, and use them together with polymer chemistry to fabricate the S-particles with desired architectures, shapes, dimensions, and materials with high precision. One major goal is to achieve such precision fabrication for pillars of diameter of 6 nm or smaller, which means to sub-6 nm size particles after biodegradation.

Surface Functioning for Sensing

The S-NP 100 becomes a nanosensor 200 after the surface functionalization. Surface functioning of S-NP is to modify the properties of the S-NP's surface to control the five key surface properties: surface shape, chemical bonding, surface charge, hydrophobic and hydrophilic properties, and active targeting (FIG. 12).

Surface Shape.

The shape of a nanoparticle has effects of the NP's ability to enter and exit of a biological materials, such as cell and cell nuclei. One embodiment is to change S-NP shape after the fabrication as needed to a desired shape. The methods of changing the shape including coating a polymer or multilayer polymer, and biodegradable materials.

Chemical Bonding.

One of the most important surface chemistry modifications is to have the linkers—the materials that link different kinds of biochemical reagent (e.g. targeting agent) onto nanoparticles. Bio-compatible block co-polymer coatings, such as polyethylene glycol (PEG), will act as a cross-linker in this proposed research. We will investigate several assembling methods, particularly two kinds. One kind is to function one end of the polymer ligands with thiol-group to form strong bonding to the nanoparticles' gold surface, and the other end of the linker with NHS-group to form strong covalent bond to the primary amine-site on the antibodies or proteins. We will also try to replace the thiol-end with silane so that the cross-linker can be efficiently linked to dielectric surface such as silica. The other method is using bio-affinity reactions such as avidin-biotin bonding. Other linkers are discussed in the section of molecular adhesion layer.

Surface Charges and Wetting Properties.

The physicochemical characteristics of a polymeric nanoparticle such as surface charge and functional groups can affect its uptake by the cells. For phagocytic system, it is well accepted that positively charged nanoparticles have a higher rate of cell uptake compared to neutral or negatively charged formulations (due to the negatively charged character of the cell plasma membrane). The coating on nanoparticles for positive charge is generally based on (or coated with) cationic polymers (e.g. the most widely used being the polysaccharide chitosan). In addition, we need to give further consideration to the surface wetting properties of the coating material, because for biocompatible materials that facilitate biodegradation, it is preferred that the coating is hydrophilic and water soluble. We will choose PEG and PGA as the positive and negative surface, respectively. Both materials are water soluble, non-toxic, biodegradable and have long been used to passivate colloid gold nanoparticle to facilitate both permeation and retention in body [13, 14]. We will test the effect of charge on the S-nanoparticle's biodistribution and clearance on both cellular level and organism level.

Effect of Active Targeting.

We will use active targeting method, where we further enhance the delivery specificity by conjugation of targeting ligands to the surface of nanoparticles. These ligands can include antibodies, engineered antibody fragments, proteins, peptides, small molecules, and DNA or RNA aptamers. We will study the effect of active targeting on both cellular level and organism level.

Molecular Adhesion Layer

The capture agents for the target analytes are immobilized either directly on S-NPs 100 or through a molecular adhesion/spacer layer (MAL) 160. As shown in FIG. 6, S-NP 100 comprises a molecular adhesion layer 160 that covers at least a part of the metal surfaces of the underlying S-NP. The molecular adhesion layer has two purposes. First, the molecular adhesion layer acts a spacer. For optimal fluorescence, the light-emitting labels (e.g., fluorophores) cannot be too close to the metal surface because non-radiation processes would quench fluorescence. Nor can the light-emitting labels be too far from the metal surface because it would reduce amplification. Ideally, the light-emitting labels should be at an optimum distance from the metal surface. Second, the molecular adhesion layer provides a good adhesion to attach capture agent onto the S-NP. The good adhesion is achieved by having reactive groups in the molecules of the molecular adhesion layer, which have a high affinity to the capture agent on one side and to the S-NPs on the other side.

The molecular adhesion layer (MAL) 160 can have many different configurations, including (a) a self-assembled monolayer (SAM) of cross-link molecules, (b) a multi-molecular layers thin film, (c) a combination of (a) and (b), and (d) a capture agent itself.

In the embodiment of MAL (a), where the molecular adhesion layer 160 is a self-assembled monolayer (SAM) of cross-link molecules or ligands, each molecule for the SAM comprises of three parts: (i) head group, which has a specific chemical affinity to the S-NP's surface, (ii) terminal group, which has a specific affinity to the capture agent, and (iii) molecule chain, which is a long series of molecules that link the head group and terminal group, and its length (which determines the average spacing between the metal to the capture agent) can affect the light amplification of the S-NP. Such a SAM is illustrated in FIG. 3.

In many embodiments, the head group attached to the metal surface belongs to the thiol group, e.t., —SH. Other alternatives for head groups that attach to metal surface are, carboxylic acid (—COOH), amine (C═N), selenol (—SeH), or phosphane (—P). Other head groups, e.g. silane (—SiO), can be used if a monolayer is to be coated on dielectric materials or semiconductors, e.g., silicon.

In many embodiments, the terminal groups can comprise a variety of capture agent-reactive groups, including, but not limited to, N-hydroxysuccinimidyl ester, sulfo-N-hydroxysuccinimidyl ester, a halo-substituted phenol ester, pentafluorophenol ester, a nitro-substituted phenol ester, an anhydride, isocyanate, isothiocyanate, an imidoester, maleimide, iodoacetyl, hydrazide, an aldehyde, or an epoxide. Other suitable groups are known in the art and may be described in, e.g., Hermanson, “Bioconjugate Techniques” Academic Press, 2nd Ed., 2008. The terminal groups can be chemically attached to the molecule chain after they are assembled to the S-NP surface, or synthesized together with the molecule chain before they are assembled on the surface.

Other terminal groups are Carboxyl —COOH groups (activated with EDC/NHS to form covalent binding with —NH2 on the ligand); Amine, —NH2, group (forming covalent binding with —COOH on the ligand via amide bond activated by EDC/NHS); Epoxy, Reacted with the —NH2 (the ligand without the need of a cross-linker); Aldehyde, (Reacted with the —NH2 on the ligand without the need of a cross-linker); Thiol, —SH, (link to —NH2 on the ligand through SMCC-like bioconjugation approach); and Glutathione, (GHS) (Ideal for capture of the GST-tagged proteins.

The molecular chain can be carbon chains, their lengths can be adjusted to change the distance between the light emitting label to the metal for optimizing the optical signal. In one embodiment, as will be described in greater detail in example section, the SAM layer is dithiobis(succinimidyl undecanoate), whose head group is —SH that binds to gold surface through sulfer-gold bond, and terminal group is NHS-ester that bind to the primary amine sites of the capture agent, and the molecule alkane chain with length of 1.7 nm.

In many embodiments, the molecule chains that link head groups and terminal groups are alkane chain, which is composed of only hydrogen and carbon atoms, with all bonds are single bonds, and the carbon atoms are not joined in cyclic structures but instead form a simple linear chain. Other alternatives for molecule chain can be ligands that are from polymers such as poly(ethylene glycol) (PEG), Poly(lactic acid) (PLA), etc. The molecule chains are chemically non-reactive to neither the metal surface that the head groups attach to, nor the capture agent that the terminal groups attach to. The chain length, which determines the distance of analyte to the S-NP's surface, can be optimized in order to achieve the maximum signal amplification. As will be described in greater detail below, the molecule chains may have a thickness of, e.g., 0.5 nm to 50 nm.

The molecular adhesion layer used in the subject nanosensor may be composed of a self-assembled monolayer (SAM) that is strongly attached to the metal at one side (via, e.g., a sulfur atom) and that terminates a capture-agent-reactive group, e.g., an amine-reactive group, a thiol-reactive group, a hydroxyl-reactive group, an imidazolyl-reactive group and a guanidinyl-reactive group, at the other (exterior) side. The monolayer may have a hydrophobic or hydrophilic surface. The most commonly used capture-agent reactive groups are NHS (which is amine-reactive) and maleimide (which is sulfhydrl-reactive), although many others may be used.

In some embodiments, the molecular adhesion layer may be a self-assembled monolayer of an alkanethiol (see, e.g., Kato Journal of Physical Chemistry 2002 106: 9655-9658), poly(ethylene)glycol thiol (see, e.g., Shenoy et al Int. J. Nanomedicine. 2006 1: 51-57), an aromatic thiol or some other chain that terminates in the thiol.

Thiol groups may be used because (a) the thiol sulfur interacts with gold and other metals to form a bond that is both strong and stable bond (see, e.g., Nuzzo et al J. Am. Chem. Soc. 1987 109:2358-2368) and (b) van der Waals forces cause the alkane and other chains to stack, which causes a SAM to organize spontaneously (see, e.g., Love et al. Chem. Rev. 2006 105:1103-1169). Further, the terminal group is available for either direct attachment to the capture molecule or for further chemical modifications.

Alkanethiol may be used in some embodiments. It has been estimated that there are 4×10¹⁴ alkanethiol molecules/cm² in a packed monolayer of alkanethiol (Nuzzo et al, J. Am. Chem. Soc. 1987 109:733-740), which approximately corresponds to an alkanethiol bond to every gold atom on the underlying surface. Self-assembled monolayers composed of alkanethiol can be generated by soaking the gold substrate in an alkanethiol solution (see, e.g., Lee et al Anal. Chem. 2006 78: 6504-6510). Gold is capable of reacting with both reduced alkanethiols (—SH groups) and alkyldisulfides (—S—S—) (see, e.g., Love et al Chem. Rev. 2005 105:1103-1169).

Once a self-assembled monolayer of poly(ethylene)glycol thiol or alkanethiol has been produced, a large number of strategies can be employed to link a capture to the self-assembled monolayer. In one embodiment, a capture agent such as streptavidin (SA) can be attached to the SAM to immobilize biotinylated capture agents.

In one embodiment, streptavidin (SA) itself can be use as a functional group (e.g. terminal group) the SAM to crosslink capture agent molecules that have high binding affinity to SA, such as biotinylated molecules, including peptides, oligonucleotides, proteins and sugars.

The functional group of avidin, streptavidin have a high affinity to the biotin group to form avidin-biotin. Such high affinity makes avidin/streptavidin serve well as a functional group and the biotin group as complementary functional group binding. Such functional group can be in binding the molecular adhesion layer to the S-NP, in binding between molecular adhesion layer and the capature agent, and in binding a light emitting lable to the secondary capture agent. In one embodiment, a molecular adhesion layer containing thiol-reactive groups may be made by linking a gold surface to an amine-terminated SAM, and further modifying the amine groups using sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC) to yield a maleimide-activated surface. Maleimide-activated surfaces are reactive thiol groups and can be used to link to capture agents that contain thiol- (e.g., cysteine) groups.

In another embodiment, a molecular adhesion layer containing an amine-reactive group (N-hydroxl succinimide (NHS)) can be produced by, e.g., by soaking the gold substrate in a 1-10 mM solution of succinimidyl alkanedisulfides such as dithiobis-sulfosuccinimidylpropionate (DSP) or dithiobis(succinimidyl undecanoate) (see, e.g., Peelen et al J. Proteome Res. 2006 5:1580-1585 and Storri et al Biosens. Bioelectron. 1998 13: 347-357).

In another embodiment, a molecular adhesion layer containing an amine-reactive group (NHS) may be produced using carboxyl-terminated SAM such as 12-carboxy-1-undecanethiol. In this case, the surface of the SAM may be linked to the NHS in the presence of 1-ethyl-3(3dimethylaminopropyl)carbodiimide HCl (EDC) to yield an inter-mediate which forms stable amide bonds with primary amines (see, e.g., Johnsson et al Anal. Biochem. 1001 198: 268-277).

In another embodiment, a molecular adhesion layer may contain Protein A which binds with high affinity to Fc region of IgGs, other immunoglobulin form, e.g., IgE.

In another embodiment, an imidazole group (which is also reactive with amines) may be added by reacting a carboxyl-terminated SAM with 1,1′-carbonyldiimidazole (CDI).

In further embodiments, aldehyde-terminated alkanethiol monolayers can be used to immobilize both proteins and amine-terminated DNA oligonucleotides, and his-tagged fusion proteins can be immobilized on nitrilotriacetic (NTA)-modified gold surfaces.

Thiol-reactive groups can link to synthetic DNA and RNA oligonucleotides, including aptamers, which can be readily synthesized commercially with a thiol terminus. Thiol-reactive groups can also link to proteins that contain a cysteine groups, e.g., antibodies. Thiolated molecules can be attached to maleimide-modified surfaces (see, e.g., Smith et al Langmuir 2002 19: 1486-1492). For in certain cases, one may use an amino acid spacer (e.g., Ser-Gly-Ser-Gly) inserted after a terminal Cys, which improves the amount of binding relative peptides that lacking spacers. For oligonucleotides, an alkane spacer can be used. Carbohydrates synthesized to contain with terminal thiols can be been tethered to gold in the same way.

Amine-reactive groups can form bonds with primary amines, such as the free amine on lysine residues. In addition to proteins, amine-reactive surfaces can be used to immobilize other biomolecules, including peptides containing lysine residues and oligonucleotides synthesized with an amine terminus.

In the embodiment of MAL (b), in which the molecular adhesion layer 160 is a multi-molecular layer thin film, the molecules may be coated on the S-NP through physical adsorption or strong binding. In one example, protein A can be coated over the entire or partial areas of the surface of S-NP surface, in which case the protein A can be deposited through physical adsorption process and has a thickness of 4 nm to 5 nm. In another example, the layer may be a thin film of a polymer such as polyethylene glycol (PEG), which has a functional head group on one end, e.g., thiol (—SH). The functioned PEG molecule layer forms a strong bond to S-NP's surface. The thickness of PEG molecule layer can be tuned by changing the PEG polymer chain length. Another example is an amorphous SiO2 thin film, which is attached to the surface of the S-NP using physical or chemical deposition methods, e.g., evaporation, sputtering, sol-gel method. The thickness of the SiO2 thin film can be precisely controlled during the deposition.

In the embodiment of MAL (c), where the molecular adhesion layer 160 is a combination of a multi-molecular layer thin film and a SAM, the SAM layer may be deposited first, followed by a multi-molecular layer.

In one example, the molecular adhesion layer may contain a monolayer of streptavidin first, followed by other layers of molecules that have high binding affinity to streptavidin, such as biotin, biotinylated molecules, including peptides, oligonucleotides, proteins, and sugars.

In one example, the molecular adhesion layer, may contain a SAM layer dithiobis(succinimidyl undecanoate) (DSU) and a Protein A layer. The DSU SAM layer binds to S-NP's metal surface through sulfer-gold bond, and has a terminal group of NHS-ester that binds to the primary amine sites on Protein A. In a particular case, capture antibodies bond to such bilayer of protein A on top of DSU through their Fc region. The protein A can ensure the orientation of antibodies for better capture efficiency.

In the embodiment of MAL (d), where the molecular adhesion layer 160 is a capture agent itself, the capture agent has a headgroup that have a high affinity to the metal or pillar sidewall of the subject S-NP. One of the common headgroup is thiol-reactive group. Thiol-reactive groups can link to synthetic DNA and RNA oligonucleotides, including aptamers, which can be readily synthesized commercially with a thiol terminus. Thiol-reactive groups can also link to proteins that contain a cysteine groups, e.g., antibodies. Another example where the MAL itself is used as the capture agent is a layer of antibody fragments, e.g., half-IgG, Fab, F(ab′)2, Fc. The antibody fragments bond to metal surface directly through the thiol-endopeptidase located in the hinge region. This embodiment is illustrated in FIG. 8. In this embodiment, the nucleic acid comprises a headgroup that binds directly the S-NP. The remainder of the steps are performed as described in FIG. 7.

The thickness of molecular adhesion layer should be in the range of 0.5 nm to 50 nm, e.g., 1 nm to 20 nm. The thickness of the molecular adhesion layer can be optimized to the particular application by, e.g., increasing or decreasing the length of the linker (the alkane or poly(ethylene glycol) chain) of the SAM used. Assuming each bond in the linker is 0.1 nM to 0.15 nM, then an optimal SAM may contain a polymeric linker of 5 to 50 carbon atoms, e.g., 10 to 20 carbon atoms in certain cases.

A nanosensor may be made by attaching capture agents to the molecular adhesion layer via a reaction between the capture agent and a capture-agent reactive group on the surface of the molecular adhesion layer.

Capture agents can be attached to the molecular adhesion layer via any convenient method such as those discussed above. In many cases, a capture agent may be attached to the molecular adhesion layer via a high-affinity strong interactions such as those between biotin and streptavidin. Because streptavidin is a protein, streptavidin can be linked to the surface of the molecular adhesion layer using any of the amine-reactive methods described above. Biotinylated capture agents can be immobilized by spotting them onto the streptavidin. In other embodiments, a capture agent can be attached to the molecular adhesion layer via a reaction that forms a strong bond, e.g., a reaction between an amine group in a lysine residue of a protein or an aminated oligonucleotide with an NHS ester to produce an amide bond between the capture agent and the molecular adhesion layer. In other embodiment, a capture agent can be strongly attached to the molecular adhesion layer via a reaction between a sulfhydryl group in a cysteine residue of a protein or a sulfhydrl-oligonucleotide with a sulfhydryl-reactive maleimide on the surface of the molecular adhesion layer. Protocols for linking capture agents to various reactive groups are well known in the art.

In one embodiment, capture agent can be nucleic acid to capture proteins, or capture agent can be proteins that capture nucleic acid, e.g., DNA, RNA. Nucleic acid can bind to proteins through sequence-specific (tight) or non-sequence specific (loose) bond.

In certain instances, a subject S-NP may be fabricated using the method: (a) patterning at least one pillar on a top surface of a substrate; (b) depositing a metallic material layer of the top surface; (c) allowing the metallic material deposited on the pillar tops to form a disc, the metallic material deposited on the pillar feet to form a metallic back plane, and the metallic material deposited on the sidewall to form at least one metallic dot structure; and, as described above, (d) depositing a molecular adhesion layer on top of the deposited metallic material, wherein the molecular adhesion layer covers at least a part of the metallic dot structure, the metal disc, and/or the metallic back plane, and wherein the exterior surface of the molecular adhesion layer comprises a capture agent-reactive group.

Furthermore, the patterning in (a) include a direct imprinting (embossing) of a material, which can be dielectric or semiconductor in electric property, and can be polymers or polymers formed by curing of monomers or oligomers, or amorphous inorganic materials. The material can be a thin film with a thickness from 10 nanometer to 10 millimeter, or multilayer materials with a substrate. The imprinting (i.e. embossing) means to have mold with a structure on its surface, and press the mold into the material to be imprinted to for an inverse of the structure in the material. The substrates or the top imprinted layers can be a plastic (i.e. polymers), e.g. polystyring (PS), Poly(methyl methacrylate) (PMMA), Polyethylene terephthalate (PET), other acrylics, and alike. The imprinting may be done by roll to roll technology using a roller imprinter. Such process has a great economic advantage and hence lowering the cost.

Sensing Systems

Also provided is a system comprising a subject nanosensor, a holder for the nanosensor, an excitation source that induces a light signal from a label (i.e. light emitting label); and a reader (e.g., a photodetector, a CCD camera, a CMOS camera, a spectrometer or an imaging device capable of producing a two dimensional spectral map of a surface of the nanosensor) adapted to read the light signal. As would be apparent, the system may also has electronics, computer system, software, and other hardware that amplify, filter, regulate, control and store the electrical signals from the reader, and control the reader and sample holder positions. The sample holder position can be move in one or all three orthogonal directions to allow the reader to scan the light signal from different locations of the sample.

The excitation source may be (a) a light source, e.g., a laser of a wavelength suitable for exciting a particular fluorophore, and a lamp or a light emitting diode with a light filter for wavelength selection; or (b) a power source for providing an electrical current to excite light out of the nanosensor (which may be employed when an electrochemiluminescent label is used).

In particular cases, laser-line pass filter filters out light whose wavelength is different from the laser, and the long wavelength pass filter will only allow the light emanate from the optically detectable label to pass through. Since different fluorescence labels absorb light in different spectral range, the fluorescence label should be chosen to match its peak absorption wavelength to the laser excitation wavelength in order to achieve optimum quantum efficiency. In many embodiments, the light signal emanating from the fluorescence label on the nanosensors are at a wavelength of at least 20 nm higher than the laser wavelength. Thus the nanosensor's plasmonic resonance should be tuned to cover the fluorescence label's absorption peak, emission peak and laser excitation wavelength. In some embodiments, the excitation and fluorescence wavelength range can be from 100 nm to 20,000 nm. The preferred range is from 300 nm to 1200 nm. The 600-850 nm range is preferable due to low background noise.

It is apparent there are other ways to achieve the functions of light excitation and reading.

As would be apparent from the above, certain nanosensors may be implemented in a multi-well format. In these embodiments, the stage can move moved so that reader can read a light signal from each of the wells of the multi-well plate, independently.

Applications in Chemical and Biological Sensing and Assay Methods

The functionalized S-particles can be used as biological and chemical sensing, including detection of biological and chemical markers, such proteins, DNAs, RNAs, and other organic and inorganic molecules, in single cells, tissue, and in-vivo for human and animals, and diagnosis.

Here diagnosis means to assess the condition of certain disease or condition by quantitative detection of certain biomarkers or biomolecules. Such diagnosis by S-NPs include detection of proteins, nucleic acids, micro-organisms (virus, bacteria, etc.) and mall molecules (hormones, etc.).

The methods of diagnosis by S-NPs include In vitro detection, fluorescence based detection, homogeneous fluorescence immunoassay (Alpha-LISA), flow-cytometery based detection, colorimetric detection, bio-bar-code Assay, SERS-based detection, SERS label homogeneous immunoassay, multiplex SERS label, in vivo detection, fluorescence Imaging (e.g. tumor cells), optical tomography and MRI.

The subject nanosensor may be used to detect analytes in a sample. This method may comprise: (a) contacting a sample comprising an analyte with a nanosensor under conditions suitable for specific binding of an analyte in the sample with the capture agent; and (b) reading an optically detectable signal from the nanosensor, wherein the optically detectable signal indicates that the analyte is bound to the capture agent. In the above step (a), before the bonding to the capture agent, the analyte may be labeled with a light-emitting label or not labeled (also referred as labeled directly or indirectly). In embodiments in which an analyte is no labeled with a light-emitting label before the bonding, the analyte, after the bonding to the capture agent, may be bound to a second capture agent (i.e. detection agent) (e.g., a secondary antibody or another nucleic acid) that is itself optically labeled, labeled secondary capture agent or labeled detection agent, (such process is also referred as indirectly labeling of an analyte). In a sensing using indirectly labeling, the labeled secondary capture agents unbounded to analytes are removed before the above reading step (b). In a sensing using directly labeling, the optical labels unbounded to analytes are removed before the above reading step (b).

In reading the light emitting labels on the assay, an excitation (photo, electro, chemical or combination of them) are applied to light emitting label, and the properties of light including intensity, wavelength, and location are detected.

In certain embodiments, the method comprises attaching a capture agent to the molecular adhesion layer of a subject S-NP to produce a nanosensor, wherein the attaching is done via a chemical reaction of the capture agent with the capture agent-reactive group in the molecules on the molecular adhesion layer, as described above. Next, the method comprises contacting a sample containing a target-analyte with the nanosensor and the contacting is done under conditions suitable for specific binding and the target-analyte specifically binds to the capture agent. After this step, the method comprises removing any target-analytes that are not bound to the capture agent (e.g., by washing the surface of the nanosensor in binding buffer); Then detection agent conjugated with optical detectable label is added to detect the target-analyte. After removing the detection agent that are not bound to the target-analyte, The S-NP can then be used, with a reading system, to read a light signal (e.g., light at a wavelength that is in the range of 300 nm to 1200 nm) from detection agent that remain bound to the nanosensor. As would be apparent, the method further comprises labeling the target analytes with a light-emitting label. This can be done either prior to or after the contacting step, i.e., after the analytes are bound to the capture agent. In certain embodiments, analytes are labeled before they are contacted with the nanosensor. In other embodiment, the analytes are labeled after they are bound to the capture agents of the nanosensor. Further, as mentioned above, the analyte may be labeled directly (in which case the analyte may be strongly linked to a light-emitting label at the beginning of the method), or labeled indirectly (i.e., by binding the target analytes to a second capture agent, e.g., a secondary antibody that is labeled or a labeled nucleic acid, that specifically binds to the target analyte and that is linked to a light-emitting label). In some embodiments, the method may comprise blocking the nanosensor prior to the contacting step (b), thereby preventing non-specific binding of the capture agents to non-target analytes.

The suitable conditions for the specific binding and the target-analyte specifically binds to the capture agent, include proper temperature, time, solution pH level, ambient light level, humidity, chemical reagent concentration, antigen-antibody ratio, etc.

In certain embodiments, a nucleic acid capture agent can be used to capture a protein analyte (e.g., a DNA or RNA binding protein). In alternative embodiments, the protein capture agent (e.g., a DNA or RNA binding protein) can be used to capture a nucleic acid analyte.

The sample may be a liquid sample and, in certain embodiments, the sample may be a clinical sample derived from cells, tissues, or bodily fluids. Bodily fluids of interest include but are not limited to, amniotic fluid, aqueous humour, vitreous humour, blood (e.g., whole blood, fractionated blood, plasma, serum, etc.), breast milk, cerebrospinal fluid (CSF), cerumen (earwax), chyle, chime, endolymph, perilymph, feces, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, sweat, synovial fluid, tears, vomit, urine and exhaled condensate.

Some of the steps of an assay are shown in FIGS. 7 and 8. General methods for methods for molecular interactions between capture agents and their binding partners (including analytes) are well known in the art (see, e.g., Harlow et al, Antibodies: A Laboratory Manual, First Edition (1988) Cold spring Harbor, N.Y.; Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995). The methods shown in FIGS. 4 and 5 are exemplary; the methods described in those figures are not the only ways of performing an assay.

Some of the steps of an exemplary antibody binding assay are shown in FIG. 5. In this assay, S-NP 100 is linked to an antibody in accordance with the methods described above to produce a nanosensor 200 that comprises antibodies 202 that are linked to the molecular adhesion layer of the S-NP. After nanosensor 200 has been produced, the nanosensor is contacted with a sample containing a target analyte (e.g., a target protein) under conditions suitable for specific binding. The antibodies 202 specifically bind to target analyte 204 in the sample. After unbound analytes have been washed from the nanosensor, the nanosensor is contacted with a secondary antibody 206 that is labeled with a light-emitting label 208 under conditions suitable for specific binding. After unbound secondary antibodies have been removed from the nanosensor, the nanosensor may be read to identify and/or quantify the amount of analyte 204 in the initial sample.

Some of the steps of an exemplary nucleic acid binding assay are shown in FIGS. 6 and 7. In this assay, S-NP 100 is linked to a nucleic acid, e.g., an oligonucleotide in accordance with the methods described above to produce a nanosensor 300 that comprises nucleic acid molecules 302 that are linked to the molecular adhesion layer. After nanosensor 300 has been produced, the nanosensor is contacted with a sample containing target nucleic acid 304 under conditions suitable for specific hybridization of target nucleic acid 304 to the nucleic acid capture agents 302. Nucleic acid capture agents 304 specifically binds to target nucleic acid 304 in the sample. After unbound nucleic acids have been washed from the nanosensor, the nanosensor is contacted with a secondary nucleic acid 306 that is labeled with a light-emitting label 308 under conditions for specific hybridization. After unbound secondary nucleic acids have been removed from the nanosensor, the nanosensor may be read to identify and/or quantify the amount of nucleic acid 304 in the initial sample.

One example of an enhanced DNA hybridization assay that can be performed using a subject device is a sandwich hybridization assay. The capture DNA is a single strand DNA functioned with thiol at its 3′-end The detection DNA is a single strand DNA functioned with a fluorescence label e.g., IRDye800CW at its 3′-end. Both the capture and detection DNA has a length of 20 bp. They are synthesized with different sequences to form complementary binding to a targeted DNA at different region. First the capture DNA is immobilized on the S-NP's metal surface through sulfur-gold reaction. Then targeted DNA is added to the S-NP to be captured by the capture DNA. Finally the fluorescence labeled detection DNA is added to the S-NP to detect the immobilized targeted DNA. After washing off the unbound detection DNA, the fluorescence signal emanate from the S-NPs' surface is measured for the detection and quantification of targeted DNA molecules.

In the embodiments shown in FIGS. 5 and 6, bound analyte can be detected using a secondary capture agent (i.e. the “detection agent”) may be conjugated to a fluorophore or an enzyme that catalyzes the synthesis of a chromogenic compound that can be detected visually or using an imaging system. In one embodiment, horseradish peroxidase (HRP) may be used, which can convert chromogenic substrates (e.g., TMB, DAB, or ABTS) into colored products, or, alternatively, produce a luminescent product when chemiluminescent substrates are used. In particular embodiments, the light signal produced by the label has a wavelength that is in the range of 300 nm to 900 nm). In certain embodiments, the label may be electrochemiluminescent and, as such, a light signal can be produced by supplying a current to the sensor.

In some embodiments, the secondary capture agent (i.e. the detection agent), e.g., the secondary antibody or secondary nucleic acid, may be linked to a fluorophore, e.g., xanthene dyes, e.g. fluorescein and rhodamine dyes, such as fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (commonly known by the abbreviations FAM and F), 6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 6-carboxy-4′, 5′-dichloro-2′, 7′-dimethoxyfluorescein (JOE or J), N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA or T), 6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G⁵ or G⁵), 6-carboxyrhodamine-6G (R6G⁶ or G⁶), and rhodamine 110; cyanine dyes, e.g. Cy3, Cy5 and Cy7 dyes; coumarins, e.g umbelliferone; benzimide dyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, e.g. cyanine dyes such as Cy3, Cy5, etc; BODIPY dyes and quinoline dyes. Specific fluorophores of interest that are commonly used in subject applications include: Pyrene, Coumarin, Diethylaminocoumarin, FAM, Fluorescein Chlorotriazinyl, Fluorescein, R110, Eosin, JOE, R6G, Tetramethylrhodamine, TAMRA, Lissamine, ROX, Napthofluorescein, Texas Red, Napthofluorescein, Cy3, and Cy5, IRDye800, IRDye800CW, Alexa 790, Dylight 800, etc.

The primary and secondary capture agents should bind to the target analyte with highly-specific affinity. However, the primary and secondary capture agents cannot be the molecule because they need to bind to different sites in the antigen. One example is the anti-human beta amyloid capture antibody 6E10 and detection G210, in which case 6E10 binds only to the 10^(th) amine site on human beta amyloids peptide while G210 binds only to the 40^(th) amine site. Capture agent and secondary capture agent do not react to each other. Another example uses rabbit anti-human IgG as capture antibody and donkey anti-human IgG as detection antibody. Since the capture and detection agents are derived from different host species, they do not react with each other.

Methods for labeling proteins, e.g., secondary antibodies, and nucleic acids with fluorophores are well known in the art. Chemiluminescent labels include acridinium esters and sulfonamides, luminol and isoluminol; electrochemiluminescent labels include ruthenium (II) chelates, and others are known.

Applications

The subject methods and compositions find use in a variety applications, where such applications are generally analyte detection applications in which the presence of a particular analyte in a given sample is detected at least qualitatively, if not quantitatively. Protocols for carrying out analyte detection assays are well known to those of skill in the art and need not be described in great detail here. Generally, the sample suspected of comprising an analyte of interest is contacted with the surface of a subject nanosensor under conditions sufficient for the analyte to bind to its respective capture agent that is tethered to the sensor. The capture agent has highly specific affinity for the targeted molecules of interest. This affinity can be antigen-antibody reaction where antibodies bind to specific epitope on the antigen, or a DNA/RNA or DNA/RNA hybridization reaction that is sequence-specific between two or more complementary strands of nucleic acids. Thus, if the analyte of interest is present in the sample, it likely binds to the sensor at the site of the capture agent and a complex is formed on the sensor surface. Namely, the captured analytes are immobilized at the sensor surface. After removing the unbounded analytes, the presence of this binding complex on the surface of the sensor (i.e. the immobilized analytes of interest) is then detected, e.g., using a labeled secondary capture agent.

Specific analyte detection applications of interest include hybridization assays in which the nucleic acid capture agents are employed and protein binding assays in which polypeptides, e.g., antibodies, are employed. In these assays, a sample is first prepared and following sample preparation, the sample is contacted with a subject nanosensor under specific binding conditions, whereby complexes are formed between target nucleic acids or polypeptides (or other molecules) that are complementary to capture agents attached to the sensor surface.

In one embodiment, the capture oligonucleotide is synthesized single strand DNA of 20-100 bases length, that is thiolated at one end. These molecules are immobilized on the S-NPs' surface to capture the targeted single-strand DNA (which may be at least 50 bp length) that has a sequence that is complementary to the immobilized capture DNA. After the hybridization reaction, a detection single strand DNA (which can be of 20-100 bp in length) whose sequence are complementary to the targeted DNA's unoccupied nucleic acid is added to hybridize with the target. The detection DNA has its one end conjugated to a fluorescence label, whose emission wavelength are within the plasmonic resonance of the S-NP. Therefore by detecting the fluorescence emission emanate from the S-NPs' surface, the targeted single strand DNA can be accurately detected and quantified. The length for capture and detection DNA determine the melting temperature (nucleotide strands will separate above melting temperature), the extent of misparing (the longer the strand, the lower the misparing). One of the concerns of choosing the length for complementary binding depends on the needs to minimize misparing while keeping the melting temperature as high as possible. In addition, the total length of the hybridization length is determined in order to achieve optimum signal amplification.

A subject sensor may be employed in a method of diagnosing a disease or condition, comprising: (a) obtaining a liquid sample from a patient suspected of having the disease or condition, (b) contacting the sample with a subject nanosensor, wherein the capture agent of the nanosensor specifically binds to a biomarker for the disease and wherein the contacting is done under conditions suitable for specific binding of the biomarker with the capture agent; (c) removing any biomarker that is not bound to the capture agent; and (d) reading a light signal from biomarker that remain bound to the nanosensor, wherein a light signal indicates that the patient has the disease or condition, wherein the method further comprises labeling the biomarker with a light-emitting label, either prior to or after it is bound to the capture agent. As will be described in greater detail below, the patient may suspected of having cancer and the antibody binds to a cancer biomarker. In other embodiments, the patient is suspected of having a neurological disorder and the antibody binds to a biomarker for the neurological disorder.

The applications of the subject sensor include, but not limited to, (a) the detection, purification and quantification of chemical compounds or biomolecules that correlates with the stage of certain diseases, e.g., infectious and parasitic disease, injuries, cardiovascular disease, cancer, mental disorders, neuropsychiatric disorders and organic diseases, e.g., pulmonary diseases, renal diseases, (b) the detection, purification and quantification of microorganism, e.g., virus, fungus and bacteria from environment, e.g., water, soil, or biological samples, e.g., tissues, bodily fluids, (c) the detection, quantification of chemical compounds or biological samples that pose hazard to food safety or national security, e.g. toxic waste, anthrax, (d) quantification of vital parameters in medical or physiological monitor, e.g., glucose, blood oxygen level, total blood count, (e) the detection and quantification of specific DNA or RNA from biosamples, e.g., cells, viruses, bodily fluids, (f) the sequencing and comparing of genetic sequences in DNA in the chromosomes and mitochondria for genome analysis or (g) to detect reaction products, e.g., during synthesis or purification of pharmaceuticals.

The detection can be carried out in various sample matrix, such as cells, tissues, bodily fluids, and stool. Bodily fluids of interest include but are not limited to, amniotic fluid, aqueous humour, vitreous humour, blood (e.g., whole blood, fractionated blood, plasma, serum, etc.), breast milk, cerebrospinal fluid (CSF), cerumen (earwax), chyle, chime, endolymph, perilymph, feces, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, sweat, synovial fluid, tears, vomit, urine and exhaled condensate.

In some embodiments, a subject biosensor can be used diagnose a pathogen infection by detecting a target nucleic acid from a pathogen in a sample. The target nucleic acid may be, for example, from a virus that is selected from the group comprising human immunodeficiency virus 1 and 2 (HIV-1 and HIV-2), human T-cell leukaemia virus and 2 (HTLV-1 and HTLV-2), respiratory syncytial virus (RSV), adenovirus, hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), human papillomavirus (HPV), varicella zoster virus (VZV), cytomegalovirus (CMV), herpes-simplex virus 1 and 2 (HSV-1 and HSV-2), human herpesvirus 8 (HHV-8, also known as Kaposi sarcoma herpesvirus) and flaviviruses, including yellow fever virus, dengue virus, Japanese encephalitis virus and West Nile virus. The present invention is not, however, limited to the detection of DNA sequences from the aforementioned viruses, but can be applied without any problem to other pathogens important in veterinary and/or human medicine.

Human papillomaviruses (HPV) are further subdivided on the basis of their DNA sequence homology into more than 70 different types. These types cause different diseases. HPV types 1, 2, 3, 4, 7, 10 and 26-29 cause benign warts. HPV types 5, 8, 9, 12, 14, 15, 17 and 19-25 and 46-50 cause lesions in patients with a weakened immune system. Types 6, 11, 34, 39, 41-44 and 51-55 cause benign acuminate warts on the mucosae of the genital region and of the respiratory tract. HPV types 16 and 18 are of special medical interest, as they cause epithelial dysplasias of the genital mucosa and are associated with a high proportion of the invasive carcinomas of the cervix, vagina, vulva and anal canal. Integration of the DNA of the human papillomavirus is considered to be decisive in the carcinogenesis of cervical cancer. Human papillomaviruses can be detected for example from the DNA sequence of their capsid proteins L1 and L2. Accordingly, the method of the present invention is especially suitable for the detection of DNA sequences of HPV types 16 and/or 18 in tissue samples, for assessing the risk of development of carcinoma.

In some cases, the nanosensor may be employed to detect a biomarker that is present at a low concentration. For example, the nanosensor may be used to detect cancer antigens in a readily accessible bodily fluids (e.g., blood, saliva, urine, tears, etc.), to detect biomarkers for tissue-specific diseases in a readily accessible bodily fluid (e.g., a biomarkers for a neurological disorder (e.g., Alzheimer's antigens)), to detect infections (particularly detection of low titer latent viruses, e.g., HIV), to detect fetal antigens in maternal blood, and for detection of exogenous compounds (e.g., drugs or pollutants) in a subject's bloodstream, for example.

The following table provides a list of protein biomarkers that can be detected using the subject nanosensor (when used in conjunction with an appropriate monoclonal antibody), and their associated diseases. One potential source of the biomarker (e.g., “CSF”; cerebrospinal fluid) is also indicated in the table. In many cases, the subject biosensor can detect those biomarkers in a different bodily fluid to that indicated. For example, biomarkers that are found in CSF can be identified in urine, blood or saliva, for example.

Marker disease Aβ42, amyloid beta-protein (CSF) Alzheimer's disease. fetuin-A (CSF) multiple sclerosis. tau (CSF) niemann-pick type C. secretogranin II (CSF) bipolar disorder. prion protein (CSF) Alzheimer disease, prion disease Cytokines (CSF) HIV-associated neurocognitive disorders Alpha-synuclein (CSF) parkinsonian disorders (neuordegenerative disorders) tau protein (CSF) parkinsonian disorders neurofilament light chain (CSF) axonal degeneration parkin (CSF) neuordegenerative disorders PTEN induced putative kinase 1 (CSF) neuordegenerative disorders DJ-1 (CSF) neuordegenerative disorders leucine-rich repeat kinase 2 (CSF) neuordegenerative disorders mutated ATP13A2 (CSF) Kufor-Rakeb disease Apo H (CSF) parkinson disease (PD) ceruloplasmin (CSF) PD Peroxisome proliferator-activated receptor PD gamma coactivator-1 alpha (PGC-1α)(CSF) transthyretin (CSF) CSF rhinorrhea (nasal surgery samples) Vitamin D-binding Protein (CSF) Multiple Sclerosis Progression proapoptotic kinase R (PKR) and its AD phosphorylated PKR (pPKR) (CSF) CXCL13 (CSF) multiple sclerosis IL-12p40, CXCL13 and IL-8 (CSF) intrathecal inflammation Dkk-3 (semen) prostate cancer p14 endocan fragment (blood) Sepsis: Endocan, specifically secreted by activated-pulmonary vascular endothelial cells, is thought to play a key role in the control of the lung inflammatory reaction. Serum (blood) neuromyelitis optica ACE2 (blood) cardiovascular disease autoantibody to CD25 (blood) early diagnosis of esophageal squamous cell carcinoma hTERT (blood) lung cancer CAI25 (MUC 16) (blood) lung cancer VEGF (blood) lung cancer sIL-2 (blood) lung cancer Osteopontin (blood) lung cancer Human epididymis protein 4 (HE4) (blood) ovarian cancer Alpha-Fetal Protein (blood) pregnancy Albumin (urine) diabetics albumin (urine) uria albuminuria microalbuminuria kidney leaks AFP (urine) mirror fetal AFP levels neutrophil gelatinase-associated lipocalin (NGAL) Acute kidney injury (urine) interleukin 18 (IL-18) (urine) Acute kidney injury Kidney Injury Molecule -1 (KIM-1) (urine) Acute kidney injury Liver Fatty Acid Binding Protein (L-FABP) (urine) Acute kidney injury LMP1 (saliva) Epstein-Barr virus oncoprotein (nasopharyngeal carcinomas) BARF1 (saliva) Epstein-Barr virus oncoprotein (nasopharyngeal carcinomas) IL-8 (saliva) oral cancer biomarker carcinoembryonic antigen (CEA) (saliva) oral or salivary malignant tumors BRAF, CCNI, EGRF, FGF19, FRS2, GREB1, and Lung cancer LZTS1 (saliva) alpha-amylase (saliva) cardiovascular disease carcinoembryonic antigen (saliva) Malignant tumors of the oral cavity CA 125 (saliva) Ovarian cancer IL8 (saliva) spinalcellular carcinoma. thioredoxin (saliva) spinalcellular carcinoma. beta-2 microglobulin levels - monitor activity of HIV the virus (saliva) tumor necrosis factor-alpha receptors - monitor HIV activity of the virus (saliva) CA15-3 (saliva) breast cancer

As noted above, a subject nanosensor can be used to detect nucleic acid in a sample. A subject nanosensor may be employed in a variety of drug discovery and research applications in addition to the diagnostic applications described above. For example, a subject nanosensor may be employed in a variety of applications that include, but are not limited to, diagnosis or monitoring of a disease or condition (where the presence of an nucleic acid provides a biomarker for the disease or condition), discovery of drug targets (where, e.g., an nucleic acid is differentially expressed in a disease or condition and may be targeted for drug therapy), drug screening (where the effects of a drug are monitored by assessing the level of an nucleic acid), determining drug susceptibility (where drug susceptibility is associated with a particular profile of nucleic acids) and basic research (where is it desirable to identify the presence a nucleic acid in a sample, or, in certain embodiments, the relative levels of a particular nucleic acids in two or more samples).

In certain embodiments, relative levels of nucleic acids in two or more different nucleic acid samples may be obtained using the above methods, and compared. In these embodiments, the results obtained from the above-described methods are usually normalized to the total amount of nucleic acids in the sample (e.g., constitutive RNAs), and compared. This may be done by comparing ratios, or by any other means. In particular embodiments, the nucleic acid profiles of two or more different samples may be compared to identify nucleic acids that are associated with a particular disease or condition.

In some examples, the different samples may consist of an “experimental” sample, i.e., a sample of interest, and a “control” sample to which the experimental sample may be compared. In many embodiments, the different samples are pairs of cell types or fractions thereof, one cell type being a cell type of interest, e.g., an abnormal cell, and the other a control, e.g., normal, cell. If two fractions of cells are compared, the fractions are usually the same fraction from each of the two cells. In certain embodiments, however, two fractions of the same cell may be compared. Exemplary cell type pairs include, for example, cells isolated from a tissue biopsy (e.g., from a tissue having a disease such as colon, breast, prostate, lung, skin cancer, or infected with a pathogen etc.) and normal cells from the same tissue, usually from the same patient; cells grown in tissue culture that are immortal (e.g., cells with a proliferative mutation or an immortalizing transgene), infected with a pathogen, or treated (e.g., with environmental or chemical agents such as peptides, hormones, altered temperature, growth condition, physical stress, cellular transformation, etc.), and a normal cell (e.g., a cell that is otherwise identical to the experimental cell except that it is not immortal, infected, or treated, etc.); a cell isolated from a mammal with a cancer, a disease, a geriatric mammal, or a mammal exposed to a condition, and a cell from a mammal of the same species, preferably from the same family, that is healthy or young; and differentiated cells and non-differentiated cells from the same mammal (e.g., one cell being the progenitor of the other in a mammal, for example). In one embodiment, cells of different types, e.g., neuronal and non-neuronal cells, or cells of different status (e.g., before and after a stimulus on the cells) may be employed. In another embodiment of the invention, the experimental material is cells susceptible to infection by a pathogen such as a virus, e.g., human immunodeficiency virus (HIV), etc., and the control material is cells resistant to infection by the pathogen. In another embodiment of the invention, the sample pair is represented by undifferentiated cells, e.g., stem cells, and differentiated cells.

Although the foregoing embodiments have been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the above teachings that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

EXAMPLES

Various S-NP particles have been fabricated by Nano-PrinTED and Dip-print and tested for enhancements of both SERS and fluorescence (tested with light wavelength of −800 nm). Exemplary results are given below.

For the S-NP structure with light resonance absorption around 800 nm wavelength, the disks have a round shape of diameter of 70 nm, the top metallic (gold) disk thickness is 15 nm, the spacer (silicon dioxide) thickness is 20 nm, and the bottom metallic (gold) disk thickness is 15 nm, the self-assembled gold dots diameter is around 10 nm, and the adhesion layers between the disks are titanium of a thickness of 0.5 nm.

In fabrication, first, the dense nanostructured protrusions template with was patterned on 4 inch substrate by nanoimprint lithography and reactive ion etching (RIE). The pillar height or etching depth was precisely controlled by RIE (FIG. 15a ). The mold can be a daughter mold duplicated by nanoimprint from a master mold fabricated with interference lithography, e-beam lithography, sphere lithography and others. Second, the multiple depositions: a release layer, metal layers (e.g. gold), adhesion layers (e.g. titanium), dielectric layers (e.g. silicon dioxide) were deposited in a sequence onto the protrusions template in a normal direction to the surface by evaporation as shown in FIG. 15b . Guided by the protrusions, the materials deposited on the top formed DS-NP arrays, and at the pillar foot, a multi-layer nano-hole backplane was also formed, the two are not connected. Third, transfer-print S-NPs to another substrate (FIG. 15c ), presents the schematic of the S-NPs array that has been transferred onto another substrate. Before transferring, a thin layer of buffer layer around 50-100 nm was spinned onto the substrate served as an adhesion layer. The transferring process was taken under low pressure (e.g. 50 PSI) and room temperature, thus not damaged the substrate nor D2-Particle arrays. Then the template peeled off the S-NPs from the templates. And fourth, solvent was used to dissolve the buffer layer and released the S-NPs into the solutions to make S-NPs (FIG. 15d ).

FIG. 13 shows the scanning electron microscopy (SEMs) of (a) double-metal-disk and single dielectric (D-particle); (b) triple-metal (or magnetic) dielectric-nanoparticle (TS-NP); (c) D-particle after the self-perfection by liquefaction (SPEL) to change the shape of 2 metal disks; (d) D-particles array on the substrate after the template lift-off.

FIG. 14. shows the scanning electron microscopy (SEMs) of (a) D-particles array on the substrate after the transfer printing. (b) D-particles exfoliated into solution.

FIG. 15 shows the Nano-PrinTED (nanoprint by templated exfoliateable deposition) fabrication of S-NP at each step. Top row: Schematic. And bottom row: scanning electron microscope (SEM) of experimental results. (a) Pillar template fabricated by lithography (e.g. NIL); (b) Multiple deposition and self-assembly to form D2-particles; (c) transfer-print DPs to another substrate; (d) put in solution. (e-h), SEM images.

As shown in FIG. 16, Nano-PrinTED and Dip-print have far better precision in controlling the NP structure dimensions (including the size and shape of each individual components, their spacing, and final particle). (a) SEM picture of D2-P before release and (b) Measured size distribution. Measured size variation of D2-particle fabricated by Nano-PrinTED (<5%) is 3 fold less than AuNP manufactured by chemical synthesis (>15%).

FIG. 17 shows the measurements of extinction spectrum of D2-particles with SiO2 layer thickness from 5 nm to 30 nm and constant Au layer thicknesses of 20 nm. Plasmonic resonant peak wavelengths redshift with increasing SiO2 layer thickness.

FIG. 18 shows the simulation of the size of nanoparticles with different architectures required for the same resonant wavelength at 800 nm. It clearly shows that S-NP has much smaller particle sized than conventional metallic sphere and disks for a given resonant wavelength.

As shown in FIG. 19, (a) Measured Surface Enhanced Raman Spectroscopy (SERS) signal of BPE, and (b) fluorescence signal of IR-800 dye with single D2-particle and gold nanoparticle. A single D2-particle has a SERS/Fluorescence enhancement over 100/30 fold higher than a single gold nanoparticle of similar diameter. The sophisticated architectures of PDS-NPs allow simultaneously improving of all three key factors for plasmonic enhancement and hence a large final enhancement. In PDS-NPs, the metallic disks (25-60 nm diameter) create antenna for good absorption of excitation light and radiation of the generated optical signal, while the smaller gaps (between the disks or additional nanodots) and sharp edges offer large local field enhancements. 

1. A nanoparticle that enhances the interaction of the nanoparticle and/or a molecule/material deposited on the surface of the nanoparticle with light, comprising a pair of stacked metallic disks separated by a non-metallic spacer, wherein: (a) the dimensions of the disks and spacer are smaller than the wavelength of the light; and (b) the nanoparticle enhance the light interaction at least three times greater than that an individual metallic disk.
 2. The nanoparticle of claim 1, wherein the light interaction includes light absorption, light scattering, light reflection, and light radiation.
 3. The nanoparticle of claim 1, wherein the light interaction includes Raman scattering, color production, and luminescence that includes fluorescence, electroluminescence, chemiluminescence, and electrochemiluminescence.
 4. The nanoparticle of claim 1, wherein the light interaction comprises interactions of light with a materials or molecule that is deposited on the nanoparticle.
 5. The nanoparticle of claim 4, wherein the molecules are analytes that have been captured on the surface of the nanoparticle.
 6. The nanoparticle of claim 4, wherein the analytes are selected from proteins, peptides, DNA, RNA, nucleic acid, small molecules, cells, and nanoparticles with different shape.
 7. The nanoparticle of claim 1, wherein the nanoparticle further comprises two masking layers covering the exterior surfaces of the metallic disks but a portion of the edges of the disks.
 8. The nanoparticle of claim 1, wherein the nanoparticle further comprises a magnetic or magnezable disk that can be attracted to a magnet.
 9. The nanoparticle of claim 8, wherein the magnetic or magnezable disk has a thickness in the range of 5 to 50 nm.
 10. The nanoparticle of claim 1, wherein the nanoparticle further comprises at least one metallic nano-dot on the edge of the spacer and/or the metallic disk.
 11. The nanoparticle of claim 10, wherein the nano-dots have a diameter in the range of 5 nm to 15 nm.
 12. The nanoparticle of claim 1, wherein the disks have the shape selected from round, polygonal, pyramidal, elliptical, elongated bar shaped, or any combination thereof.
 13. The nanoparticle of claim 1, wherein the metallic and the spacer have the same and similar lateral dimension.
 14. The nanoparticle of claim 1, wherein, for light enhancement in 800 nm wavelength and near by region, the disks have a significantly round shape of diameter from 30 nm to 100 nm, the top metallic disk thickness is from 5 nm to 30 nm, the spacer thickness is from 2 to 30 nm, and the bottom metallic disk thickness is from 5 nm to 30 nm.
 15. The nanoparticle of claim 1, wherein the nanoparticle further comprises a magnetic or magnezible disk, that can be attracted to a magnet.
 16. The nanoparticle of claim 1, wherein the stacked metallic disks are made of the same or different materials.
 17. The nanoparticle of claim 1, wherein the material for the metallic disks is selected from the group consisting of gold, silver, copper, aluminum, alloys thereof, and combinations thereof.
 18. The nanodevice of claim 1, wherein the distance between the pair of the metallic disk is in the range of 0.1 nm to 20 nm, for the light wavelength of 800 nm and around.
 19. The nanodevice of claim 1, wherein the lateral dimension of said metallic disc is in the range from 5 nm to 150 nm.
 20. The nanodevice of claim 1, wherein said metallic disc and the metallic back plane are spaced by a distance in the range of 0.1 nm to 60 nm.
 21. The nanodevice of claim 1, wherein said at least one metallic dot structure has dimensions in the range of 1 nm to 25 nm.
 22. The nanodevice of claim 1, wherein the distance between said metallic dot structure and said metallic disc, and the distance between said metallic dot structure and said metallic backplane is in the range of 0.5 nm to 50 nm. 23-30. (canceled) 